摘要
目的
检测TMP和联合As_2O_3后对HL-60细胞增殖、分化的影响，并探讨其分子机制。建立SCID小鼠-人AML模型，体内验证TMP减毒增效功能，并探讨其机制。
方法
1.TMP抑制HL-60细胞增殖和诱导分化的作用。MTT法检测TMP对HL-60细胞增殖影响；NBT还原能力实验检测TMP作用下HL-60细胞向粒系分化时功能成熟程度，并筛选出TMP最佳诱导分化浓度；瑞氏染色观察细胞形态学变化；流式细胞仪检测细胞表面分化抗原CD11b、CD14表达率；流式细胞仪检测HL-60细胞周期分布；RT-PCR、Western blot检测细胞周期相关mRNA和蛋白：c-myc、p27、CDK2和cyclinE1表达。
2. TMP联合As_2O_3对HL-60细胞增殖的影响。采用HL-60和ECV-304两种细胞株，分为空白对照组、TMP组、As_2O_3组、TMP联合As_2O_3组。MTT法检测HL-60细胞增殖变化；RT-PCR测定HL-60细胞VEGF mRNA表达；ELISA检测HL-60细胞分泌VEGF蛋白的差异。台盼蓝染色计数绘制ECV-304细胞生长曲线；流式细胞仪检测ECV-304细胞早期凋亡率；倒置显微镜观察ECV-304细胞贴壁情况。
3.TMP联合As_2O_3对HL-60细胞诱导分化的作用。分组同前。MTT实验检测HL-60细胞增殖变化；瑞氏染色观察细胞形态学变化；NBT还原能力实验检测HL-60细胞向粒系分化时功能成熟程度；流式细胞仪检测HL-60细胞表面分化抗原CD11b、CD14表达率；流式细胞仪检测细胞周期分布；用RT-PCR、Western blot检测c-myc、p27、CDK2和cyclinE1mRNA和蛋白表达。
4.建立SCID小鼠-人AML模型，对TMP减毒增敏功能进行体内实验验证。建模：在给予SCID小鼠300cGy (~(60)Co source)放射24h内，尾静脉注射HL-60细胞（1×10~6个）建立模型。免疫组织化学检测小鼠骨髓单个核细胞CD33蛋白表达阳性率和HE染色观察白血病细胞侵润组织情况共同来鉴定建模是否成功。分组：建模后的小鼠随机分为4组：患病组（不予药物治疗）、单用TMP组（200mg/kg TMP腹腔注射，Qd×21天）、单用As_2O_3组（3mg/kg As_2O_3腹腔注射，Qd×21天）、TMP联合As_2O_3组（200mg/kg TMP腹腔注射+3mg/kg As_2O_3腹腔注射，Qd×21天）。各组小鼠濒死前麻醉后处死。取小鼠血清，检测肝肾功能；ELISA检测血清中VEGF蛋白分泌量；免疫荧光检测小鼠骨髓微小血管密度；免疫组织化学检测小鼠骨髓单个核细胞CD33、HIF-1α蛋白表达。
结果
1.随着药物浓度的增加，TMP对HL-60细胞的生长的抑制作用逐渐增强；在低浓度(200-300μg/ml) TMP作用下NBT阳性细胞率明显升高。300μg/ml TMP处理HL-60细胞不同时间后，CD11b阳性表达率升高呈时间依赖性（p＜0.01），CD14阳性表达率无统计学差异；细胞被阻滞于G0/G1期；c-myc mRNA和蛋白表达水平明显下调（p＜0.01），p27mRNA和蛋白表达水平显著上升（p＜0.01），cyclinE1和CDK2mRAN水平变化无统计学意义，但其蛋白表达水平则显著下降（p＜0.01），呈时间依赖性。
2.在TMP+As_2O_3组HL-60细胞生长抑制率与空白对照组或单用As_2O_3组相比显著升高（p＜0.05），HL-60细胞VEGF mRNA和蛋白表达在联合处理后明显减少。在TMP+As_2O_3组ECV-304细胞早期凋亡率与空白对照组或单用As_2O_3组相比显著升高（p＜0.01）；经TMP和As_2O_3单独处理后均可抑制ECV-304细胞对培养瓶壁的粘附能力，联合作用后效果明显增强。
3.在TMP联合As_2O_3组HL-60细胞NBT还原率和粒系分化抗原CD11b表达率显著增高（p＜0.01）；油镜下观察到细胞在形态学上亦趋于成熟；p27mRNA和蛋白表达水平都较对照组显著上升（p＜0.05），c-myc mRNA和蛋白表达水平均明显下调（p＜0.01），cyclinE1mRNA表达水平下降（p＜0.05），其蛋白表达水平下降更明显（p＜0.01），CDK2mRAN表达变化轻微，但其蛋白表达水平显著下调（p＜0.01）。
4.预处理后SCID小鼠注射HL-60细胞后其骨髓单个核细胞CD33呈强阳性表达，肝、脾、肾、骨髓及转移瘤组织病理切片显示有大量AML白血病细胞浸润，证实SCID小鼠-人AML模型建立成功。
各组白血病SCID小鼠生存时间存在显著差异，各治疗组小鼠生命延长率分别是：单用TMP组29.63％，单用As_2O_3组50％，TMP联合As_2O_3组74.07％。AST在患病组显著少于As_2O_3组（p＜0.05）,AST、CREA在联合用药组显著少于As_2O_3组（p＜0.05）。CD33蛋白在患病组骨髓单个核细胞中表达呈强阳性，在单用As_2O_3组、单用TMP组呈中度表达，在联合用药组呈低表达。HIF–la蛋白在患病组呈强阳性表达，在单用As_2O_3组呈中度表达，在单用TMP组呈低表达，联合用药组表达阴性。
在联合用药组VEGF蛋白分泌量也较单用药物组和患病组显著降低。患病组白血病SCID小鼠骨髓微血管的血管形态幼稚，血管腔狭小，血管管壁结构紊乱；治疗组白血病SCID小鼠骨髓微血管管腔较对照增大，血管结构较完整。各治疗组小鼠骨髓MVD显著地低于患病组（p＜0.05），联合用药组明显低于其余各组（p＜0.05），单药治疗组之间骨髓MVD计数无差异。
结论
1.小剂量TMP能诱导HL-60细胞分化，使细胞周期阻滞于G0/G1期，其机制可能是c-myc表达的下调，p27表达的增加，在蛋白水平抑制与cyclin E-CDK2复合物的结合，使细胞周期停滞于G0/G1期。
2.TMP联合As_2O_3后抑制HL-60细胞增殖作用增强。其机制可能是一方面HL-60细胞VEGF mRNA合成和蛋白分泌减少，通过VEGF自分泌途径促进白血病细胞凋亡，通过VEGF旁分泌途径中抑制血管内皮细胞ECV-304的增殖；另一方面抑制内皮细胞ECV-304的增殖与转移，阻断血管新生的重要环节。
3.小剂量TMP能增强小剂量As_2O_3诱导分化的作用，以粒系分化为主。其机制可能是TMP通过上调p27mRNA和蛋白表达，下调c-mycmRNA和蛋白表达，抑制cyclin E-CDK2复合物蛋白激酶活性，阻滞细胞由G1期向S期过渡，促使HL-60细胞分化成熟。
4. TMP能增强As_2O_3对SCID小鼠-人AML模型的治疗作用，明显改善小鼠的肝肾功能。其增效的机制可能是TMP下调白血病模型小鼠体内HIF-1a和VEGF的表达，小鼠骨髓MVD减少，微小血管结构趋向正常化，使AML细胞凋亡率升高。该实验填补了TMP在抗血管生成作用动物模型的空白。
Objectives
Human acute promyelocytic leukemia HL-60cells and umbilical veinendothelial ECV-304cells were selected in the study. On the basis of thefunction of resisting anoxia and antiangiogenesis in TMP, we detected theeffect of the tetramethylpyrazin（eTMP）alone or in combination of arsenictrioxide（As_2O_3） on proliferation and differentiation in human acutepromyelocytic leukemia HL-60cells, and investigated the possiblemechanism based on the molecular of VEGF and H1F-1a. As well toinvestigate TMP could potentiate As_2O_3activity against leukemia further invivo, we establish the SCID mice model suffering from human AML.
Methods
1. Effects of TMP on proliferation and differentiation in HL-60cells.The alteration in the cell proliferation of TMP（concentration of100、200、300、400、600、800、1000μg/ml）was detected by MTT experiment; Thefunctional maturity of differentiation in HL-60cells was determined by NBTtest, and filter the suitable concentration in the following experiments; Cell morphology was observed by Wright’s staining; Flow cytometry was used todetect the expression of CD11b/CD14and the cell cycle distribution inHL-60cells. The cell cycle related mRNA and protein such as c-myc、p27、CDK2and cyclinE1were detected by RT-PCR and Western blot.
2. Effects of TMP alone or in combination of As_2O_3on proliferation inHL-60cells. HL-60cells and ECV-304cells were used in this partialexperiment. There were four groups: control group, TMP treated group,As_2O_3treated group and TMP+As_2O_3treated group. The alteration in theproliferation was detected by MTT; RT-PCR and ELISA were assayed toexamine the expression of VEGF mRNA and protein; Trypan blue stainingwas down to draw the growth curves of ECV-304cells; Flow cytometry wasused to determine the early apoptosis of ECV-304cells; the adhesion degreeof ECV-304cell was observed by inverted microscopy.
3. Effects of TMP alone or in combination of As_2O_3on proliferation inHL-60cells. There were four groups: control group, TMP treated group,As_2O_3treated group and TMP+As_2O_3treated group. The alteration in thecell proliferation was detected by MTT experiment; The functional maturityof differentiation in HL-60cells was determined by NBT test, and filters thesuitable concentration in the following experiments; Cell morphology wasobserved by Wright’s staining; Flow cytometry was used to detect theexpression of CD11b/CD14and the cell cycle distribution in HL-60cells.The cell cycle related mRNA and protein such as c-m yc、 p27、CDK2and cyclinE1were detected by RT-PCR and Western blot assay.
4.As well to investigate TMP could potentiate As_2O_3activity againstleukemia further in vivo, we establish the SCID mice model suffering fromhuman AML. HL-60cells (1×10~6) were injected into SCID mice via a tailvein following irradiated at300cGy (~(60)Co source)24h earlier. Theexpression of CD33in bone marrow mononuclear cells detected byimmunohistochemistry and the infiltrated organization observed by HEstaining were used to identify whether the model is successfully established.The SCID mice models were randomly divided into four groups: controlgroup, TMP treated group (200mg/kg, i.p., Qd×21d), As_2O_3treated group(3mg/kg, i.p., Qd×21d), TMP+As_2O_3treated group (200mg/kgTMP+3mg/kg As_2O_3, i.p., Qd×21d). The mice were put to death afteranesthesia at the death hour. The liver and kidney function was detected aftergotten their serum; the expression of the secreted VEGF protein wasexamined by ELISA; Bone Marrow Microvessel density (BMMVD) wasscored using immunofluorescence; the expression of CD33and HIF-1αprotein in bone marrow mononuclear cells detected byimmunohistochemistry.
Results
1. TMP inhibited the proliferation of HL-60cells in a dose andtime-dependent manner. TMP induced the differentiation of HL-60cells inthe relatively low concentration (200-300μg/ml). After exposed to TMP in the300μg/ml concentration, the cells were blocked in the G0/G1cell cycleprogression. The mRNA and protein expression of c-myc were graduallydecreased, while remarkably up-regulated in the expression of p27. Therewere no obviously changes in the mRAN expression of CDK2and cyclinE1;however the protein expressions of them were extantly declined in atime-dependent manner.
2. TMP can obviously potentiate As_2O_3ability of inhibitingproliferation in HL-60and ECV-304cells. The mRNA expression andsecretory protein of VEGF remarkably declined after exposure to thecombination treatment. Both of TMP and As_2O_3can induce the earlyapoptosis and adhesion ability of ECV-304cells, and these effects wereenhanced when treated combined.
3. Combination treatments had synergistic effects on the proliferativeinhibition rates. The rates were increased gradually after the combinationtreatment, much higher than those treated by corresponding As_2O_3alone.The cells exhibited characteristics of mature granulocytes and a higherNBT-reducing ability, being a2.6-fold increase in the rate of NBT-positiveratio of HL-60cells within the As_2O_3treatment versus almost a13-foldincrease in the TMP+As_2O_3group. Cells treated with both TMP and As_2O_3expressed far more CD11b antigens, almost2-fold compared with controlgroup. The mRNA and protein expression of c-myc and cyclinE1weregradually decreased, while remarkably up-regulated in the expression of p27. There were no obviously changes in the mRAN expression of CDK2;however the protein expressions of them were extantly declined.
4. The high expression of CD33protein in mice bone marrowmononuclear cells and an abundant of leukemic cells appeared in micetissues of liver, kidney, spleen, bone marrow and transplanted tumordemonstrated that SCID mice model suffering from human AML hadalready established. The survival time in each experimental group hadsignificant difference. The prolonged rates of survival time were29.63％inTMP group,50％in As_2O_3group and74.07％in combined treated grouprespectively. The liver and kidney function dramatically got better incombined treated group compared with As_2O_3group. CD33protein inBMMC were strongtly positive expressed in control group, moderatelyexpressed in TMP or As_2O_3group, and low expressed in combined group.HIF-la protein were strongtly positive expressed in control group,moderately expressed in As_2O_3group, low expressed in TMP group, andnegative expressed in combined group. VEGF protein in serum incombined group was dramatically decreased. In control group the lumen inmice bone marrow microvascular got much smaller and the microvascularhad many small branches. The bone MVD in each mice treated group wasdramatically decreased than that in control group, and that of which incombined group was decreased than that in other group. The bone MVD ineach treated alone group had no significant difference.
Conclusions
1. Small doses of TMP can induce differentiations of HL-60cells, andblock the cells in G0/G1cell cycle progression. The mechanism waspossibly by down-regulating the c-myc mRNA and protein expression,up-regulating the p27mRNA and protein expression and inhibiting thecombination of the protein cyclin E and CDK2.
2. TMP potentiates As_2O_3inhibiting proliferation ability in HL-60cellsvia the pathways of VEGF autocrine and paracrine. The study preliminarilydemonstrated that antiangiogenesis is the mechanism of TMP alone or incombination of As_2O_3treated AML.
3. Small doses of TMP potentiate As_2O_3-induced differentiation ofHL-60cells, possibly by regulating the expression and activity of G0/G1phase-arresting molecules. Up-regulating the expression of p27caninhibiting the combination of the protein cyclin E and CDK2, conductive tothe DNA synthesis and the down-regulation of relative genes such as c-mycneeded in the transition of the G1→S cell cycle progression. Thecoordination and interaction of the upward and downward genes induceddifferentiation of HL-60cells.
4. Injecting HL-60cells into pretreated SCID mice via a tail vein cansuccessfully establish SCID mice-human AML model. TMP can potentiateAs_2O_3anticancer ability in SCID mice suffering from AML, reduce the toxicside effect of As_2O_3in liver and kidney function, possibly by down-regulating the expression of HIF-1a and VEGF, inhibiting the growthof the bone marrow microvacular and increasing the apoptosis rate of HL-60cells.
引文
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